Although a significant number of cancers have been cured by selective chemotherapeutic agents, many remain untreatable. The latter group includes those that are intrinsically drug resistant and those that acquire resistance following chemotherapy. Because both resistant types, in general, are resistant to a number of related as well as unrelated chemotherapeutic agents, they have been designated as multidrug resistant (mdr). A significant amount of data have accumulated during the last decade characterizing the mdr phenotype. Mdr cells have increased levels of a 170 kd phosphorylated protein, p-glycoprotein, which functions in drug efflux. However, the regulation and modification of p-glycoprotein remains unknown. Additionally, some mdr cells demonstrate increased phosphorylation of other low mw cellular proteins such as the 22 kd, Ca2+ binding protein, sorcin, which too, may function in mdr. There have been some suggestions that p-glycoprotein, as well s the aforementioned low mw proteins, may be substrates for protein kinase C (PKC), and several studies have demonstrated increased levels of PKC in mdr human cancers which show increased levels of protein phosphorylation. Our laboratory has developed a rapid 2-D gel electrophoresis procedure for analyzing PKC isoforms in HL60/ADR cells (unpublished results) and identified PKC gamma as the major isoform. This correlates with our observation of increased phosphorylation of a low mw protein in these cell lines. Furthermore, we have recently obtained preliminary evidence that this phosphorylated protein may be sorcin since it reacts to sorcin antibody. The major objectives of this proposal are: (1) to characterize the PKC isozyme pattern in human mdr leukemic and breast cancer drug-resistant cell lines and, (2) to correlate these findings with the phosphorylation of p- glycoprotein, sorcin, and other cellular proteins in these cell lines. The ultimate goal is to determine if these metabolic processes can be exploited for the development of new pharmaceuticals designed to reverse the mdr phenotype. The technical approach will include standard cell culture procedures and protein extractions. Characterization of PKC isozymes will be accomplished by high-resolution two-dimensional gel electrophoresis and Western blotting, and radioactive assays for PKC activity. Phosphorylation of p- glycoprotein and sorcin and whether each serves as substrates for PKC will be determined by metabolic labeling, autoradiography, immunoprecipitation and immunoblotting.